Abstract

RNA editing is a post-transcriptional modification in which adenosine residues are converted to inosine (adenosine-to-inosine editing). Commonly used methodologies to quantify RNA editing levels involve either direct sequencing or pyrosequencing of individual cDNA clones. The limitations of these methods lead to a small number of clones characterized in comparison to the number of mRNA molecules in the original sample, thereby producing significant sampling errors and potentially erroneous conclusions. We have developed an improved method for quantifying RNA editing patterns that increases sequence analysis to an average of more than 800,000 individual cDNAs per sample, substantially increasing accuracy and sensitivity. Our method is based on the serotonin 2C receptor (5-hydroxytryptamine(2C); 5HT(2C)) transcript, an RNA editing substrate in which up to five adenosines are modified. Using a high-throughput multiplexed transcript analysis, we were able to quantify accurately the expression of twenty 5HT(2C) isoforms, each representing at least 0.25% of the total 5HT(2C) transcripts. Furthermore, this approach allowed the detection of previously unobserved changes in 5HT(2C) editing in RNA samples isolated from different inbred mouse strains and dissected brain regions, as well as editing differences in alternatively spliced 5HT(2C) variants. This approach provides a novel and efficient strategy for large-scale analyses of RNA editing and may prove to be a valuable tool for uncovering new information regarding editing patterns in specific disease states and in response to pharmacological and physiological perturbation, further elucidating the impact of 5HT(2C) RNA editing on central nervous system function.

Summary of the HTMTA sequencing strategy. A, the structure and alternative splicing of 5HT2C pre-mRNA in the exon 5–6 region is presented (top). The locations of RT-PCR primers are indicated (black arrows). Adapter sequences for paired-end sequencing on the Illumina Genome Analyzer (dark blue) were incorporated into each primer. An annealing region for a sequencing primer (light blue) and one of eight distinct barcode sequences (yellow) were incorporated into the antisense primer. The sequence of the nonedited RNA, beginning immediately after the primer sequence, is specified below the pre-mRNA RT-PCR diagram. The positions of the five edited adenosines are indicated in boldface type (sites A, B, E, C, and D), and the amino acids encoded by the nonedited (top) and fully edited transcripts (bottom) are indicated below their respective codons. The position of the 36-nt sequence obtained from read 1 is indicated between the dashed lines on both the RT-PCR and pre-mRNA diagrams. Two sequencing primers (read 1 and read 2, orange arrows) were used for paired-end sequence analysis of RT-PCR amplicons generated from 5HT2C mRNA (bottom). Each of eight unique barcode sequences incorporated into the RT-PCR amplicon for sample identification is provided in the yellow inset. B, flow chart of data-sorting criteria. Total sequence reads obtained per RNA sample from HTMTA were culled to an appropriate data set by meeting a series of inclusion criteria, including the following: 1) barcode sequences are identifiable; 2) RNA 2 or RNA 3 alternatively spliced isoforms are identifiable; 3) nucleotides at the editing site are either A or G; and 4) the sequence of read 1 precisely matches the mouse 5HT2C RNA 2 reference sequence (Supplemental Table S2). The representative example is derived from the analysis of eight distinct barcoded samples. The number of reads that fail to meet each inclusion criterion is indicated to the right, followed by the percentage of the total reads that these excluded sequences represent. The number of sequence reads meeting each criterion is indicated below the inclusion description in each rectangle.

Comparison of RNA editing patterns using HTMTA and pyrosequencing methodologies. A, quantitative analysis of site-specific 5HT2C editing in RNA isolated from five independent whole-brain mouse samples as determined by HTMTA. B, quantitative analysis of 5HT2C editing using the same RNA samples as above by pyrosequencing. The mean percentage of site-specific editing is listed below each site (±S.E.M.).